The present invention relates to glycosyl-etoposide prodrugs, a process for the preparation thereof and the use thereof in combination with functionalized tumor-specific enzyme conjugates for treating cancers, and specifically relates to 4xe2x80x2-O-glycosyl-etoposides as prodrugs which can be cleaved by the action of tumor-specific enzyme conjugates to give cytotoxic active substances, the liberated active substance being suitable, by reason of its cytostatic activity, for treating cancers.
The present invention further relates to enzyme conjugates for prodrug activation, including fusion proteins of the general formula huTuMAb-L-xcex2-Gluc, where huTuMAb is a humanized or human tumor-specific monoclonal antibody, a fragment or a derivative thereof, L is linker, and xcex2-Gluc comprises human xcex2-glucuronidase. These fusion proteins are prepared by genetic manipulation. huTuMAb ensures the specific localization of tumors, L connects the huTuMAb to xcex2-Gluc in such a way that the specific properties of the two fusion partners are not impaired, and xcex2-Gluc activates a suitable prodrug compound by elimination of glucuronic acid, where a virtually autologous system for use in humans is provided by the humanized or human fusion partners.
The combination of prodrug and tumor-specific antibody-enzyme conjugates for use as therapeutic agents is described in the specialist literature. This has entailed injection of antibodies which are directed against a particular tissue and to which a prodrug-cleaving enzyme is covalently bonded into an animal which contains the transplanted tissue, and subsequently administering a prodrug compound which can be activated by the enzyme. The prodrug is converted by the action of the antibody-enzyme conjugate, which is anchored to the tissue, into the cytotoxin which exerts a cytotoxic effect on the transplanted tissue.
A therapeutic system which contains two components, an antibody-enzyme component and a prodrug component that can be activated by the enzyme is described in WO 88/07378. In this case, the use of non-mammalian enzymes is described for the preparation of the antibody-enzyme conjugates, and that of endogenous enzymes is ruled out because of non-specific liberation of active compound. Since the exogenous enzymes are recognized by the body as foreign antigens, the use thereof is associated with the disadvantage of an immune response to these non-endogenous substances, which can result in the enzyme immobilized on the antibody being inactivated, or possibly, the entire conjugate being eliminated. In addition, in this case p-bis-N-(2-chloroethyl) amino-benzylglutamic acid and derivatives thereof are used as prodrug, and the chemical half-life thereof is only 5.3 to 16.5 hours. It is a disadvantage for a prodrug to be chemically unstable because of the side effects to be expected.
A therapeutic system which contains two components and in which the antibody-enzyme conjugate located on the tumor tissue cleaves a prodrug compound to form a cytotoxic active compound is likewise described in EPA 0302473 A2. The combined use of etoposide 4xe2x80x2-phosphate and derivatives thereof as prodrug and of antibody-immobilized alkaline phosphatases for liberating the etoposides, which is described therein, inter alia, is disadvantageous because of the presence of large amounts of endogenous alkaline phosphatases in the serum. As described in DE 38265662 A1, the etoposide 4xe2x80x2-phosphates have already been used alone as therapeutic antitumor agents, with the phosphatases present in the serum liberating the etoposide from the prodrug.
It has emerged, surprisingly, that the synthetically prepared, hitherto unobtainable compound 4xe2x80x2-O-alpha-D-glucopyranosyl-etoposide can be cleaved in vitro into etoposide and D-glucose with the enzyme alpha-glucosidase as well as a tumor-specific antibody-glucosidase conjugate.
Based on this finding, and taking into account the disadvantages, described above, of prior art combinations of prodrugs and antibody-enzyme conjugates, the object of the present invention was to prepare synthetic, enzymatically cleavable 4xe2x80x2-O-glycosyl-etoposides as well as functionalized tumor-specific enzymes to cleave them, and to test the pharmacological utility of the combination of the two components in suitable mammalian test models. This object has been achieved by preparing 4xe2x80x2-O-glycosyl-etoposides of formula I and functionalized tumor-specific enzymes of formula II which, on combined use thereof, exhibit cytostatic activity.
The invention thus relates to 4xe2x80x2-O-glycosyl-etoposides of the formula I 
in which
R1 is a methyl, benzyl or 2-thienyl group,
R2 is a hydrogen atom, an acyl or tri-C1-C4-alkylsilyl protective group,
R3 is a hydroxl group, an acyl or tri-C1-C4-alkylsilyl protective group which is bonded via an oxygen atom, an amino, acetylamino, benzyloxycarbonylamino or dimethylamino group,
R4 is a hydrogen atom or a methyl group,
R5 is a hydrogen atom, a hydroxyl group, an acyl or tri-C1-C4-alkylsilyl protective group which is bonded via an oxygen atom, or an amino, benzyloxycarbonyl-amino, azido or acetylamino group,
R6 is a hydroxyl group, an acyl or tri-C1-C4-alkylsilyl protective group which is bonded via an oxygen atom, or an amino, benzyloxycarbonylamino or azido group,
R7 is a hydrogen atom, an acyl or tri-C1-C4-alkylsilyl protective group, and
R8 is a methyl or hydroxymethyl group or an acyl protective group which is bonded via a methyleneoxy group, or a benzyloxycarbonyl group, where an acyl protective group means an acetyl, mono-, di- or trihalogenoacetyl group with halogen meaning fluorine or chlorine;
xe2x80x83and functionalized tumor-specific enzymes of the formula II
Axe2x80x94Spxe2x80x94Exe2x80x83xe2x80x83II
xe2x80x83in which
A is an antibody or one of the fragments thereof, which have specificity for a tumor-associated antigen, or is a biomolecule which accumulates in a tumor, such as EGF (epidermal growth factor), TGF-xcex1 (transforming growth factor a), PDGF (platelet derived growth factor), IGF I+II (insulin like growth factor I+II) or a+b FGF (acidic+basic fibroblast growth factor),
E is a glycosidase which is not immunogenic or is of low immunogenicity, preferably mammalian glycosidase, as xcex1-or xcex2-glucosidase, xcex1-galactosidase, xcex1- or xcex2-mannosidase, xcex1-fucosidase, N-acetyl-xcex1-galactosaminidase, N-acetyl-xcex2-/N-acetyl-xcex1-glucosaminidase or xcex2-glucuronidase,
Sp (spacer) is a polypeptide spacer or a bifunctional sulfide- or disulfide-containing group of the formula III or IV
X(S)nYxe2x80x83xe2x80x83III
X(S)nxe2x80x83xe2x80x83IV
xe2x80x83in which
X is xe2x80x94COxe2x80x94R9xe2x80x94(N-succinimido)- or xe2x80x94C(xe2x95x90R)xe2x80x94CH2xe2x80x94CH2xe2x80x94 with R9 being xe2x80x94CH2xe2x80x94CH2xe2x80x94, 1,4-cyclohexylidene, 1,3- or 1,4-phenylene or methoxycarbonyl- or chloro-1,4-phenylene and R being O or NH,
Y is xe2x80x94C(xe2x95x90R10)xe2x80x94CH2xe2x80x94CH2xe2x80x94, where R10 has the stated meaning, and
n is 1 or 2.
Preferred within the scope of the invention are compounds of the formula I in which the radicals
R1 is a methyl, benzyl or 2-thienyl group,
R2 is a hydrogen atom, an acetyl or chloroacetyl group or a tri-C1-C4-alkylsilyl protective group,
R3 is a hydroxyl group, an acetyl, chloroacetyl or tri-C1-C4-alkylsilyl protective group which is bonded via an oxygen atom, or an amino, acetylamino, benzyloxycarbonylamino or dimethylamino group,
R4 is a hydrogen atom or a methyl group,
R5 is a hydrogen atom, a hydroxyl group, or an acetyl, chloroacetyl or tri-C1-C4-alkylsilyl protective group which is bonded via an oxygen atom, or an amino, benzyloxycarbonylamino, azido or acetylamino group,
R6 is a hydroxyl group, an acetyl, chloroacetyl or tri-C1-C4-alkylsilyl protective group which is bonded via an oxygen atom, or an amino, benzyloxycarbonyl-amino or azido group,
R7 is a hydrogen atom, an acetyl, chloroacetyl or tri-C1-C4-alkylsilyl protective group, and
R8 is a methyl, hydroxymethyl, acetyloxy or chloro-acetyloxymethyl group or a benzyloxycarbonyl group.
Also preferred within the scope of the invention are functionalized tumor-specific enzyme conjugates of the formula II in which
A is an antibody or fragment thereof, which have specificity for a tumor-associated antigen, or is a biomolecule which accumulates on or in the tumor, such as EGF (epidermal growth factor), TGF-xcex1 (transforming growth factor xcex1), PDGF (platelet derived growth factor), IGF I+II (insulin like growth factor I+II), a+b FGF (acidic+basic fibro-blast growth factor),
E is a glycosidase which is not immunogenic or has low immunogenicity, preferably a mammalian glycosidase, for example an xcex1- or xcex2-glucosidase, xcex1-galactosidase, xcex1- or xcex2-mannosidase, xcex1-fucosidase, N-acetyl-xcex1-galactosaminidase, N-acetyl-xcex2-/N-acetyl-xcex1-glucosaminidase or xcex2-glucuronidase, and
Sp is a polypeptide spacer or a bifunctional disulfide-containing group of the formula III or IV
xe2x80x83in which
X is xe2x80x94COxe2x80x94R9xe2x80x94(N-succinimido)- or xe2x80x94C(xe2x95x90R10)xe2x80x94CH2xe2x80x94CH2xe2x80x94 with R9 being xe2x80x94CH2xe2x80x94CH2xe2x80x94 or 1,4-phenylene and R10 being O or NH,
Y is xe2x80x94C(xe2x95x90R10)xe2x80x94CH2xe2x80x94H2xe2x80x94, where R10 has the stated meaning, and
n is 1 or 2.
Of these preferred functionalized tumor-specific enzyme conjugates, it has been found that certain tumor-specific antibody-glucosidase conjugates, or fusion proteins, comprising a humanized or human tumor-specific monoclonal antibody, or a fragment or derivative thereof, linked to human xcex2-glucuronidase, and prepared by genetic manipulation, are particularly advantageous, because they are virtually autologous. Moreover, it has been found that the catalytic activity of 3-glucuronidase in such fusion proteins at pH 7.4 (i.e. physiological conditions) is significantly higher than that of the native enzyme when the fusion protein is bound to the antigen via the V region. Furthermore, a fusion protein with only one hinge region (see Table 4 and Example 22) can be generated by genetic manipulation in high yield because most of the product which is formed results as one band (in this case with molecular weight 125,000) and can be easily purified by affinity chromatography with anti-idiotype MAbs or anti-glucuronidase MAbs.
Accordingly, especially preferred tumor-specific enzyme conjugates of the invention are fusion proteins of the formula V
huTuMAb-L-xcex2-Glucxe2x80x83xe2x80x83V
in which
huTuMAb is a humanized or human tumor-specific monoclonal antibody or a fragment or a derivative thereof, and preferably comprises the MAbs described in EP-A1-0 388 914. The fusion proteins according to the invention particularly preferably contain the humanized MAb fragment with the VL and VH genes shown in Table 3,
L is a linker and preferably contains a hinge region of an immunoglobulin which is linked via a peptide sequence to the N-terminus of the mature enzyme, and
xcex2-Gluc is the complete amino-acid sequence of human xcex2-glucuronidase or, in the relevant gene constructs, the complete cDNA (Oshima A. et al., Proc. Natl. Acad. Sci. USA 84, (1987) 685-689).
In these fusion proteins, the huTuMAb ensures the specific localization of tumors, L connects huTuMAb to xcex2-Gluc in such a way that the specific properties of the two fusion partners are not impaired, and xcex2-Gluc activates a suitable prodrug compound by elimination of glucuronic acid, where a virtually autologous system for use in humans is provided by the humanized or human fusion partners.
Furthermore preferred are constructs with a CH1 exon and a hinge exon in the antibody part, and particularly preferred constructs are those in which these parts derive from a human IgG3 C gene. Most preferred are constructs, as described in Example 16, where the corresponding light chain of the humanized TuMab is co-expressed in order, in this way, to obtain an huTuMAb portion which is as similar as possible to the original TuMAb in the binding properties.
Furthermore, it has been found that a chemical modification of the fusion proteins, in particular partial or complete oxidation of the carbohydrate structures, preferably with subsequent reductive amination, results in an increased half-life. Enzymatic treatment of the fusion proteins according to the invention with alkaline phosphatase from, for example, bovine intestine or E. coli has in general not resulted in a significant increase in the half-life.
Accordingly, in another embodiment, the fusion proteins according to the invention are chemically modified in order to achieve an increased half-life and thus an improved localization of tumors. The fusion proteins are preferably treated with an oxidizing agent, for example periodate, which generally results in partial or complete cleavage of the carbohydrate rings and thus in an alteration in the carbohydrate structure. This alteration generally results in an increased half-life. It is furthermore advantageous to derivatize, in a second reaction step, existing aldehyde groups, for example by reductive amination. The partial or complete destruction of the aldehyde groups generally results in a reduction in possible side reactions with, for example, plasma proteins. Accordingly, it is advantageous for the fusion proteins according to the invention to be oxidized in a first reaction step, for example with periodate, and to be reductively aminated in a second reaction step, for example with ethanolamine and cyanoborohydride.
The process according to the invention for preparing a compound of the formula I, which can be degraded by glycosidase, in which
R1 is a methyl, benzyl or 2-thienyl group,
R2 is a hydrogen atom,
R3 is a hyroxyl, amino or dimethylamino group,
R4 is a hydrogen atom or a methyl group,
R5 is a hydrogen atom, a hydroxyl group, an amino or acetylamino group,
R6 is a hydroxyl group or an amino group,
R7 is a hydrogen atom, and
R8 is a methyl or hydroxymethyl group or a carboxyl group or an acyl protective group which is bonded via a methyleneoxy group, or a benzyloxycarbonyl group, where an acyl protective group means an acetyl, mono-, di- or trihalogenoaceyl group with halogen meaning fluorine or chlorine,
xe2x80x83comprises reacting, in the presence of a promoter and, where appropriate, of an acid trap or drying agent in a solvent at xe2x88x9250xc2x0 C. to 60xc2x0 C., an etoposide compound of the formula VI 
xe2x80x83in which
R is a methyl, benzyl or 2-thienyl group,
R2 is a hydrogen atom, an acyl or a tri-C1-C4-alkylsilyl protective group,
R3 is a hydroxyl group, an acyl or tri-C1-C4-alkylsilyl protective group which is bonded via oxygen, or acetylamino, benzyloxycarbonylamino or dimethylamino group, and
R4 is a hydrogen atom or a methyl group, with a carbohydrate component of the formula VII 
xe2x80x83in which
R5 is a hydrogen atom, a hydroxyl group, an acyl protective group which is bonded via an oxygen atom, or benzyloxycarbonylamino, azido or acetylamino group,
R6 is an acyl protective group which is bonded via an oxygen atom, or a benzyloxycarbonylamino or azido group,
R7 is an acyl protective group,
R8 is a methyl group, methyleneoxy-acyl protective group or a benzyloxycarbonyl group and
Z is a halogen atom, preferably fluorine, chlorine or bromine, a hydroxyl group, a tri-C1-C4-alkylsilyloxy group, or an acyl protective group which is bonded via an oxygen atom, where the acyl protective group is an acetyl, mono-, di- or trihalogeno-acetyl group, preferably with the halogen atom being fluorine or chlorine,
xe2x80x83to give a 4xe2x80x2-O-glycosyl-etoposide derivative of the formula I in which all the radicals R1 to R8 retain their meaning as defined above, and eliminating the protective groups present in these compounds by hydrogenolysis or hydrolysis, and, where appropriate, converting by means of reductive alkylation one of the resulting compounds containing amino groups into another compound of the formula I containing dimethylamino groups.
The specific procedure for this is as follows: the glycosidation of etoposide derivatives of the formula VI is carried out using functionalized carbohydrate units of the formula VII which are typically protected with acyl protective groups on the O-2, O-3, O-4 and, where appropriate, O-6 atoms. Preferred acyl protective groups are acetyl, chloroacetyl or trifluoroacetyl groups. In the case of amino sugars, the amino group is protected temporarily with the benzyloxycarbonyl group or permanently with an acetyl group. It is likewise possible to use azido sugars because they can be converted straightforwardly into amino sugars by hydrogenolysis. The carbohydrate components must be suitably functionalized at the anomeric center. Used for.this purpose are glycosyl halides, such as fluorides, chlorides or bromides, which can be prepared starting from 1-O-acyl derivatives, for example using HF, HCl, HBr or TiBr4. The glycosidation components which carry an O-acyl group or a hydroxyl group on the anomeric center are prepared by processes customary in carbohydrate chemistry.
The glycosidation of etoposides of the formula VI with carbohydrate units of the formula VII is carried out in the presence of a promoter. The promoter used when glycosyl fluorides and the 1-hydroxy or 1-acetyloxy analogs thereof are employed is BF3x ether or tri-C1-C4-alkylsilyl trifluoromethanesulfonate. The promoters used in the case of glycosyl chlorides or bromides are salts of silver or of mercury.
The glycosidation is carried out in an aprotic organic solvent such as acetone, ethyl acetate, ether, toluene, dichloromethane or dichloroethane or mixtures thereof. In order to trap the acid or water produced in the reaction, where appropriate, acid traps or drying agents such as molecular sieves or magnesium sulfate are added. The reaction temperature is in the range from xe2x88x9250xc2x0 C. to 0xc2x0 C. when glycosyl fluorides and the 1-hydroxy analogs are employed and at 0xc2x0 C. to 60xc2x0 C. when glycosyl chlorides or bromides are employed. The glycosyl etoposides produced in the reaction are deblocked by the following processes: the acyl protective groups are removed by methanolysis catalyzed by zinc (II) salts or with alkaline ion exchangers in methanol, ethanol or mixtures thereof with chloroform, dichloromethane or ether. The benzyl or benzyloxycarbonyl groups or azido groups are eliminated by hydrogenolysis with palladium on carbon or palladium/barium sulfate or, in the case of the azido group, converted into amino group. The compounds of the formula I containing amino sugars can additionally be converted into dimethylamino derivatives by reductive alkylation with formaldehyde/sodium cyanoborohydride.
To prepare the functionalized tumor-specific enzyme conjugates of the invention, the spacer (Sp) can be linked via an amino group to an enzyme and to the antibody or the biomolecule via an HS group which has been introduced or generated by cleavage of the disulfide linkage, or nucleic acid sequences which code for the parts A, Sp and E are covalently linked with the aid of molecular biological methods to result in a fusion gene, and Axe2x80x94Spxe2x80x94E is prepared by genetic engineering processes.
The coupling is carried out by processes described in the literature (A. H. Blair and T. I. Ghose, (1983) J. Immunolog. Methods 59, 129-143; T. I. Ghose et al. (1983) Methods in Enzymology, Vol. 93, 280-333). This entails initial functionalization of the enzyme via its amino group using succinimidyl N-maleimido-alkylidene-, cycloalkylidene- or arylene-1-carboxylate, where the double bond of the maleimido group enters into a reaction with the HS group of the functionalized antibody, fragment thereof or the biomolecules, with the formation of a thioether functionality.
Preparing the functionalized tumor-specific enzyme conjugates by genetic engineering processes can be carried out in a variety of ways:
A) A restriction cleavage site A is introduced by specific mutagenesis at the 3xe2x80x2 end of the CH1 exon in the gene of the heavy chain of the immunoglobulin. The same restriction cleavage site A is generated at the 5xe2x80x2 end of the oligonucleotide which codes for the oligopeptide which acts as spacer. Both restriction cleavage sites A are sited in such a way that the immunoglobulin gene can be linked to the oligonucleotide via the restriction cleavage site A without disturbing the reading frame.
A restriction cleavage site B is generated at the 3xe2x80x2 end of the oligonucleotide. This restriction cleavage site B is introduced at the site in the gene which codes for the enzyme at which the nucleic acid sequence coding for the mature protein starts. The enzyme gene is then linked via the restriction cleavage site B to the immunoglobulin gene-linked construct. The restriction cleavage sites B are sited such that the reading frame is not disturbed on linkage. The fusion gene composed of the DNA for the heavy chains of the immunoglobulin VH and CH1 linker enzyme is cloned into an expression plasmid which is suitable for expression in the eukaryotic cells and carries a selection marker.
The expression plasmid with the fusion gene is transfected together with an expression plasmid which carries the gene for the light chain belonging to the antibody into eukaryotic cells (for example myeloma cells). Selection with suitable antibiotics is carried out to isolate cell clones which contain the plasmids with the fusion gene and the gene for the light chains (transfectomas). Suitable detection methods (BioDot; ELISA) are used to identify those transfectomas which secrete the fusion protein of the formula Axe2x80x94Spxe2x80x94E composed of the MAb Fab part, linker polypeptide and enzyme.
B) A restriction cleavage site A is introduced at the 3xe2x80x2 end of the hinge exon of the gene for the heavy chains of the immunoglobulin. The restriction cleavage site A is introduced at the site in the enzyme gene at which the nucleotide sequence coding for the mature protein starts. The gene fragment of the heavy chains of the immunoglobulin with the VHxe2x80x2, CH1 and hinge exons is linked via the restriction cleavage site A to the enzyme gene.
The restriction sites A are sited such that the reading frame is not disturbed on linkage. The hinge part of the antibody functions as the polypeptide spacer in this construction. The fusion gene composed of VHxe2x80x2 CH1 hinge and enzyme gene is cloned into an expression plasmid which is suitable for expression in eukaryotic cells and carries a selection marker. The expression plasmid with the.fusion gene is transfected together with an expression plasmid which contains the light-chain gene belonging to the antibody into eukaryotic expression cells. Selection with a suitable antibiotic is followed by identification of transfectoma clones which contain the expression plasmids. Suitable detection methods (BioDot, ELISA) are used to identify those transfectoma clones which secrete the fusion protein of the formula II composed of antibody andenzyme.
It is possible to use for the preparation of the antibody-enzyme conjugates the monoclonal antibodies described in EP-A-0141079, preferably the antibodies 431/26, 250/183, 704/152 and 494/32. The specificity of the antibodies for tumor-associated antigens has already been demonstrated on animals and humans by means of immunoscintigraphy and immunohistochemistry.
The nucleotide sequence of the V genes of these monoclonal antibodies is described in German Patent Application DE-A-39099799.4.
To prepare the tumor-specific enzyme conjugates, it is possible for the enzymes which are mentioned hereinafter and from the identified source to be purified by the indicated literature procedures:
xcex1-galactosidase from human liver, Dean, K. G. and Sweeley, C. C. (1979) J. Biol. Chem. 254, 9994-10000
xcex2-glucuronidase from human liver, Ho, K. J. (1985) Biochem. Biophys. Acta 827, 197-206
xcex1-L-fucosidase from human liver, Dawson, G., Tsay, G. (1977) Arch. Biochem. Biophys. 184, 12-23
xcex1-mannosidase from human liver, Grabowski, G. A., Ikonne, J. U., Desnick, R. J. (1980) Enzyme 25, 13-25
xcex2-mannosidase from human placenta, Noeske, C., Mersmann, G. (1983) Hoppe-Seyler""s Z. Physiol. Chem. 364, 1645-1651
xcex1-glucosidase from human gastrointestinal mucosa, Asp, N.-G., Gudmand-Hoeyer, E., Christiansen, P. M., Dahlquist, A. (1974) Scand. J. Clin. Lab. Invest. 33, 239-245
xcex2-glucosidase from human liver, Daniels, L. B., Coyle, P. J., Chiao, Y.-B, Glew, R. H. (1981) J. Biol. Chem. 256, 13004-13013
xcex2-glucocerebrosidase from human placenta, Furbish, F. S., Blair, H. E., Shiloach, J., Pentcheu, P. G., Brady, R. O. (1977) Proc. Natl. Acad. Sci. USA 74, 3560-3563
xcex1-N-acetylglucosaminidase from human placenta, Rohrborn, W., von Figura, K. (1978) Hoppe-Seyler""s Z. Physiol. Chem. 359, 1353-1362
xcex2-N-acetylglucosaminidase from human amniotic membrane, Orlacchio, A., Emiliani, C., Di Renzo, G. C., Cosmi, E. V. (1986) Clin. Chim. Acta 159, 279-289
xcex1-N-acetylgalactosaminidase according to Salvayre, R., Negre, A., Maret, A., Douste-Blazy, L. (1984) Pathol. Biol. (Paris) 32, 269-284.
The glycolytic activity of the functionalized tumor-specific enzymes was determined in comparative investigations with p-nitrophenyl glycosides at the particular pH optimum.
To test the efficacy of a combined sequential use, transplanted mice were given the functionalized enzyme, then, after waiting until the plasma level of the enzyme had fallen virtually to zero, the glycosyletoposide was given and it was observed whether growth stopped and regression occurred.